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PubMed1975

Specific cross-linking of proteins S7 and L4 to ribosomal RNA, by UV irradiation of Escherichia coli ribosomal subunits.

Radioactive 30S and 50S subunits from E. coli ribosomes were irradiated with UV light, under conditions giving rise to approximately 10% cross-linking of protein to ribosomal RNA. Irradiation to levels of cross-linking higher than 10% caused unfolding of the ribosomal subunits, even in the presence of 5 mM magnesium. The specificity of the cross-linking reaction at this low level was found to be extremely high. Cross-linked RNA-protein complexes, freed from unbound protein, were treated with nuclease and then analysed on Sarkosyl gels. S7 was found to be the primary target of the cross-linking reaction in the 30S particle. This was proven by using subunits from both E. coli MRE 600 and A19, whose respective S7 species differ markedly. In the 50S particle, L4 was the primary target, although L2 was also cross-linked to a small extent. Ambiguity in the identification of L4 in the Sarkosyl system was resolved by two-dimensional electrophoresis, which was also used to demonstrate a genuine linkage to RNA in the case of both S7 and L4; proteins spots containing 32P were observed, derived from these two proteins, when subunits containing 32P-RNA were irradiated, treated with nuclease, and applied to the electrophoresis. The identities of S7, L4 and L2 were finally confirmed by Ouchterlony tests with protein-specific anti-sera.

Electrophoresis, Polyacrylamide Gel↗

PubMed1993

Heterobasidion annosum 5.8s ribosomal DNA and internal transcribed spacer sequence: rapid identification of European intersterility groups by ribosomal DNA restriction polymorphism.

Using conserved fungal ribosomal gene sequences the internal transcribed spacer (ITS) regions one and two (ITS1, ITS2) and the 5.8s ribosomal RNA gene (rRNA) of Heterobasidion annosum were amplified by the polymerase chain reaction (PCR). The nucleotide sequence was determined in three European intersterility groups (ISG-S, -F and -P). Three sequence variants of the ITS were found in ISG-S isolates. The sequence of the ITS of ISG-F differed by two residues from the major ISG-S sequence variant. The ISG-P sequence differed from ISG-S and ISG-F at 15-16 and 16 residues, respectively. Amplified intergenic spacer elements were informative for ISG fingerprinting following digestion with various 4-cutter restriction endonucleases. All differences in the restriction fragments between the ISGs were because of sequence differences in the ITS regions. The fingerprint patterns of isolates from the same intersterility group but from different European localities were identical. These results show that ribosomal DNA fingerprinting is a rapid technique to identify ISGs in Heterobasidion annosum.

Base Sequence↗

PubMed1996

Functional promoter elements common to ribosomal protein and ribosomal RNA genes in Neurospora crassa.

The promoter sequences of a cytoplasmic ribosomal protein gene (crp-2) of Neurospora crassa were identified using promoter deletion and substitution mutants. A gene-targeting strategy was used to assay the mutants in vivo. The promoter architecture of crp-2 is complex and is more similar to that of ribosomal protein genes in mouse than in Saccharomyces cerevisiae. Six regions were identified as important for transcription. These included two elements, a CG repeat and a Dde box, that are conserved in most other promoters of N. crassa ribosomal protein genes and have also been demonstrated as being required for transcription from the 40 S rRNA promoter by RNA polymerase I in vitro. The CG repeats located at -73 to -66 and between -189 and -154 were functionally redundant and increased transcription efficiency by 10- to 15- fold. The Dde boxes located at -153 to -147 and at -95 to -83 contributed 2-fold and 5-fold to transcription efficiency, respectively. An unidentified element between -254 and -190 contributed 2-fold, while a pyrimidine-rich region between -85 and -66 influenced the start point of transcription.

Base Sequence↗

PubMed1976

Release of certain ribosomal proteins from 70-S Escherichia coli ribosomes by mild ribonuclease digestion.

A method for the release of some proteins from Escherichia coli (MRE 600)ribosomes is described, avoiding extraction with denaturing reagents. High-salt-washed, 70-S ribosomes were treated with pancreatic ribonuclease which led to the release of 12 proteins of the larger ribosomal subunit. Separation of released protein was first attempted by gel filtration and by fractionation with (NH4)2SO4. The number and type of the released proteins were identified by sodium dodecyl sulphate-polyacrylamide gels and two-dimensional gel electrophoresis. The method should prove of use in the large scale purification of proteins L1, L7, and L25.

Bacterial Proteins↗

PubMed1983

Immunoelectron microscopy of ribosomes carrying a fluorescence label in a defined position. Location of proteins S17 and L6 in the ribosome of Escherichia coli.

By coupling fluorescein to a defined amino acid of a single ribosomal protein and incorporating this protein into the ribosome, we have obtained ribosomes labelled at a single, defined position. A fluorescein-specific antibody preparation was used to locate the fluorescein residues bound to the two cysteines at positions 58 and 63 of protein S17 and to the cysteine at position 86 of protein L6. This study demonstrates the advantages which accrue from the combination of electron microscopy and fluorimetry.

Antibodies↗

PubMed1991

rig encodes ribosomal protein S15. The primary structure of mammalian ribosomal protein S15.

rig, a gene originally isolated from a rat insulinoma cDNA library, codes for a basic 145 amino acid protein [( 1986) Diabetes 35, 1178-1180]. Here we show that the immunoreactivity to a monoclonal antibody against the deduced rig protein and the translation product of rig mRNA comigrated with ribosomal protein S15. The amino acid sequence of ribosomal protein S15 purified from rat liver coincided with that deduced from the nucleotide sequence of rig mRNA, but there were indications that the initiator methionine was removed and the succeeding alanyl residue was monoacetylated. From these results, we conclude that the product of rig is ribosomal protein S15.

Amino Acid Sequence↗

PubMed1995

In vivo assembly of plasmid-expressed ribosomal protein S7 of Thermus thermophilus into Escherichia coli ribosomes and conditions of its overexpression.

Researchers still have great difficulty in isolating individual ribosomal proteins from the ribosome in quantities high enough for structural research. To this end, when studying protein S7, we created an E. coli overproducer of the recombinant protein S7 of Thermus thermophilus. The vector for expression was pQE-32 having a strong promoter of E. coli phage T5 and six triplets of His at the 5'-end. This N-terminal six His tag of the fusion protein is responsible for binding to Ni-NTA-resin and allows purifying the protein in one step. The yield of the recombinant protein was 20% and more of the total cellular proteins. In addition we have shown that the recombinant thermophilic protein is incorporated in vivo into the ribosome of E. coli despite the fact that these proteins (thermophilic and mesophilic) have a rather low homology, only 52%. This fact provides a base for the system to study functions of individual proteins.

Amino Acid Sequence↗

PubMed1984

Characterization of rat ribosomal DNA. The highly repetitive sequences that flank the ribosomal RNA transcription unit are homologous and contain RNA polymerase III transcription initiation sites.

The non-transcribed spacers (NTS) of the ribosomal genes of a number of organisms have been studied and were found to contain repetitive sequences. In these studies with plasmid subclones of NTS, designated p3.4, p2.6 and p1.7, which come from both 5' and 3' flanking regions of the rat ribosomal genes, respectively, it has been determined that these sequences are found elsewhere within the genome. Southern hybridization analysis has demonstrated that the 5' and 3' NTS subclones cross-hybridize, and that the cross-hybridizing regions are synonymous with the highly repetitive regions. Sequences homologous to the rat NTS were specifically localized to both 5' and 3' flanking regions as well as to a number of the introns of cloned genes including rat serum albumin, rat alpha-fetoprotein, rat casein and human serum albumin. No hybridization was detected of the 5' NTS subclone to the human Alu sequence clone, Blur 8, or to the rodent equivalent, a clone containing Chinese hamster ovary type I and II Alu sequences. However, as reported for type II Alu sequences, the subcloned rat NTS sequences contain RNA polymerase III initiation sites and also hybridize to a number of small RNAs, but not 4.5 S or 7 S RNA. Sequence analysis of two distinct repetitive regions in p1.7 has revealed a region of alternating purine-pyrimidine nucleotides, potentially of Z DNA, and stretches of repetitive sequences. The possible roles for these repetitive sequences in recombination and in maintaining a hierarchical structure for the ribosomal genes are discussed.

Animals↗

PubMed1986

Rapid chemical probing of conformation in 16 S ribosomal RNA and 30 S ribosomal subunits using primer extension.

We have investigated in detail the higher-order structure of 16 S ribosomal RNA, both in its naked form and in 30 S ribosomal subunits. Each base in the 16 S rRNA chain has been probed using kethoxal (which reacts with guanine at N1 and N2), dimethylsulfate (which reacts with adenine at N1 and cytosine at N3) and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (which reacts with uracil at N3 and guanine at N1). The sites of reaction were identified by primer extension with reverse transcriptase using synthetic oligodeoxynucleotide primers. These results provide a detailed and rigorous experimental test of a model for 16 S rRNA secondary structure, which was derived mainly from comparative sequence analysis. Our data also provide information relevant to tertiary and quaternary structure of 16 S rRNA. Data obtained with naked 16 S rRNA show reasonably close agreement with the proposed model, and data obtained with 30 S subunits show nearly complete agreement. Apart from an apparent overall "tightening" of the structure (in which many weakly reactive bases become unreactive), assembly of the proteins with 16 S rRNA to form 30 S subunits brings about numerous local structural rearrangements, resulting in specific enhancements as well as protections. In many instances, the ribosomal proteins appear to "tune" the 16 S rRNA structure to bring it into accordance with the phylogenetically predicted model, even though the RNA on its own often seems to prefer a different structure in certain regions of the molecule. Extensive protection of conserved, unpaired adenines upon formation of 30 S subunits suggests that they play a special role in the assembly process, possibly providing signals for protein recognition.

Aldehydes↗

PubMed1983

The effect of ionic and temperature shifts used for in vitro ribosome subunit reconstitution upon the large molecular weight ribosomal ribonucleic acids.

The behaviour of purified, intact preparations of 16 S and 23 S rRNAs has been studied in their respective ribosome subunit reconstitution systems by means of sedimentation and electrophoretic analysis. Both species undergo profound conformation changes to more compact species at the temperatures and ionic conditions commonly agreed for their reconstitution into ribosome subunits. The 16 S rRNA undergoes a complete conformation change over the input range of concentration used, whereas the change for 23 S rRNA is incomplete for inputs above 1.5 mg X ml-1. Intact 23 S rRNA is also required and some preparations recommended for r-protein binding studies do not meet this requirement for reconstitution. These observations are discussed in relation to the overall effectiveness of ribosome subunit reconstitution systems.

Escherichia coli↗

PubMed1983

The in vitro and in vivo inactivation of ribosomes by virginiamycin M does not entail an alteration of 5, 16 and 23 S ribosomal RNA.

The M component of virginiamycin blocks protein synthesis by inactivating catalytically the 50 S ribosomal subunits: the in vitro interaction of 50 S with virginiamycin M, followed by removal of the antibiotic, results in a lasting damage of the particle. This enzyme-like inactivation of 50 S subunits resembles that of 30 S subunits by colicin E3, which entails the cleavage of 16 S rRNA. To explore this possibility, rRNA obtained from particles incubated in vivo and in vitro with virginiamycin M were analyzed. Electrophoretic analysis of 5, 16 and 23 S rRNA did not reveal major changes, nor did it show the appearance of additional fragments. To exclude the possibility of terminal alterations, the 5'- and 3'-extremities of these RNA were also sequenced and found unchanged. Conclusions drawn in the present work parallel those of an accompanying paper (Moureau, P., Di Giambattista, M. and Cocito, C. (1983) Biochim. Biophys. Acta 739, 164-172) describing the dissociation and reassociation of ribosomes incubated with virginiamycin M: the lasting ribosome damage by this antibiotic appears to be due to a conformational rather than to a structural alteration.

Bacillus subtilis↗

PubMed1990

Structural arrangement of the decoding site of Escherichia coli ribosomes as revealed from the data on affinity labelling of ribosomes by analogs of mRNA--derivatives of oligoribonucleotides.

Using derivatives of oligoribonucleotides bearing an active group at the 5'- or 3'-end, the affinity modification of Escherichia coli ribosomes has been investigated in model complexes imitating various steps of initiation and elongation with a different extent of approximation to the real protein-synthesizing system. The protein environment of the ribosome decoding site is determined. The S3, S4, S9, L2, L7/L12 proteins belong to the 5'-region of the decoding site, and the S5, S7, S9, L1, L16 proteins to the 3'-region. In the process of translation the template moves along the external side of the 30 S subunit, from the L1 ridge to the L7/L12 stalk. The structural arrangement of the decoding site or its nearest environment depends on the functional state of ribosomes in the process of translation.

Affinity Labels↗

PubMed1991

Mutants lacking individual ribosomal proteins as a tool to investigate ribosomal properties.

We have isolated and characterized mutants which lack one or two of sixteen of the proteins of the Escherichia coli ribosome. The mutation responsible in each case mapped close to, and probably in, the corresponding gene. A conditional lethal phenotype and a variable degree of impairment in growth was observed. The missing protein was readily restored to the organelle if E coli or other eubacterial ribosomal proteins were added to a suspension of the mutant particles. The mutants have been used to investigate the role of individual proteins in ribosome function and assembly. They have also aided in the topographic pinpointing of proteins on the surface of the organelle.

Aminoglycosides↗

PubMedDate not supplied

Crystallography of ribosomes: attempts at decorating the ribosomal surface.

Crystals of various ribosomal particles, diffracting best to 2.9 A resolution were grown. Crystallographic data were collected from shock frozen crystals with intense synchrotron radiation at cryo temperature. For obtaining phase information, monofunctional reagents were prepared from an undecagold and a tetrairidium cluster, by attaching to them chemically reactive handles, specific for sulfhydryl moieties. Heavy-atom derivatives were prepared by a specific and quantitative binding of the undecagold cluster to an exposed sulfhydryl prior to the crystallization. To create potential binding sites on the halophilic and thermophilic ribosomal particles, which yield our best and most interesting crystals, exposed reactive moieties were inserted, using genetic and chemical procedures. In order to choose the appropriate locations for these insertions, the surfaces of the ribosomal particles were mapped by direct chemical determination of exposed amino and sulfhydryl groups.

Bacteria↗

PubMed2004

Rrs1p, a ribosomal protein L11-binding protein, is required for nuclear export of the 60S pre-ribosomal subunit in Saccharomyces cerevisiae.

Rrs1p is a ribosomal protein L11-binding protein in Saccharomyces cerevisiae. We have obtained temperature-sensitive rrs1 mutants by random PCR mutagenesis. [(3)H]Methionine pulse-chase analysis reveals that the rrs1 mutations cause a defect in maturation of 25S rRNA. Ribosomal protein L25-enhanced green fluorescent protein, a reporter of the 60S ribosomal subunit, concentrates in the nucleus with enrichment in the nucleolus when the rrs1 mutants are shifted to the restrictive temperature. These results suggest that Rrs1p stays on the pre-60S particle from the early stage to very late stage of the large-subunit maturation and is required for export of 60S subunits from the nucleolus to the cytoplasm.

Active Transport, Cell Nucleus↗

PubMed1983

Immunochemical accessibility of ribosomal protein S4 in the 30 S ribosome. The interaction of S4 with S5 and S12.

The reactivity of protein S4-specific antibody preparations with 30 S ribosomal subunits and intermediates of in vitro subunit reconstitution has been characterized using a quantitative antibody binding assay. Anti-S4 antibody preparations did not react with native 30 S ribosomal subunits; however, they did react with various subunit assembly intermediates that lacked proteins S5 and S12. The inclusion of proteins S5 and S12 in reconstituted particles resulted in a large decrease in anti-S4 reactivity, and it was concluded that proteins S5 and S12 are primarily responsible for the masking of S4 antigenic determinants in the 30 S subunit. The effect of S5 and S12 on S4 accessibility is consistent with data from a variety of other approaches, suggesting that these proteins form a structural and functional domain in the small ribosomal subunit.

Antibodies, Bacterial↗

PubMed2001

Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: mature 5S rRNA is dispensable for ribosome function.

Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598-600). Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G + C gram-positive bacteria. We propose that the gene be named rnmV. The rnmV gene is nonessential. B. subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function. 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along gram-negative and gram-positive lines. A potential correlation between the presence of RNase E or RNase M5 and the single- or double-stranded nature of the predicted cleavage sites is explored.

Amino Acid Sequence↗

PubMed1992

Interaction between the two conserved single-stranded regions at the decoding site of small subunit ribosomal RNA is essential for ribosome function.

Formation of the tertiary base pair G1401:C1501, which brings together two universally present and highly sequence-conserved single-stranded segments of small subunit ribosomal RNA, is essential for ribosome function. It was previously reported that mutation of G1401 inactivated all in vitro functions of the ribosome [Cunningham et al. (1992) Biochemistry 31, 7629-7637]. Here we show that mutation of C1501 to G was equally inactivating but that the double mutant C1401:G1501 with the base pair reversed had virtually full activity for tRNA binding to the P, A, and I sites and for peptide bond formation. Initiation-dependent formation of the first peptide bond remained 70-85% inhibited, despite full 70S initiation complex formation ability as evidenced by the ability to form fMET-puromycin. These results suggest that the defect in formation of the first peptide bond lies in filling the initial A site, Ai, rather than the subsequent elongation A sites, Ae. An increased mobility around the anticodon was detected by UV cross-linking of the anticodon of P-site-bound tRNA to C1399 as well as to the expected C1400. These findings provide the first experimental evidence for the existence of the G1401:C1501 base pair and show that this base pair, located at the decoding site, is essential for function. The structural implications of tertiary base pair formation are discussed.

Base Sequence↗